ISSN 00036838, Applied Biochemistry and Microbiology, 2011, Vol. 47, No. 2, pp. 176–181. © Pleiades Publishing, Inc., 2011. 2 Original Russian Text © N.R. Meichik, N.I. Popova, Yu.I. Nikolaeva, I.P. Yermakov, A.N. Kamnev, 2011, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2011, Vol. 47, No. 2, pp. 194–200. IonExchange Properties of Cell Walls of Red Seaweed Phyllophora Crispa 2 N. R. Meichik, N. I. Popova, Yu. I. Nikolaeva, I. P. Yermakov, and A. N. Kamnev Department of Plant Physiology, Biological Faculty, Moscow State University, Moscow, 119991 Russia email: meychik@mail.ru Received June 20, 2010 Abstract—Research into ionexchange properties of cell walls isolated from thallus of red seaweed Phyllo phora crispa was carried out. Ionexchange capacity and the swelling coefficient of the red alga cell walls were estimated at various pH values (from 2 to 12) and at constant ionic strength of a solution (10 mM). It was established that behavior of cell walls as ionexchangers is caused by the presence in their matrix of two types of cationexchange groups and amino groups. The amount of the functional group of each type was esti mated, and the corresponding values of pKαwere calculated. It can be assumed that ionogenic groups with pKα ~5 are carboxyl groups of uronic acids, and ionogenic groups with pKα ~7.5 are carboxyl groups of the proteins. Intervals of pH in which cationexchange groups are ionized and can take part in exchange reac tions with cations in the environment are defined. It was found that protein was a major component of cell wall polymeric matrix because its content was 36%. DOI: 10.1134/S000368381102013X Macrophytes are the most important dominants of Black Sea biocommunities. Macro vegetation per forms the function of a major coastal area producer, strengthens coastal soils, prevents penetration of anthropogenic substances into them, provides food reserves, and is the place of spawning and shelter for fish and invertebrates. From the practical viewpoint, red algae are important specimens of macrophytes. From red algae, components of the cell wall, agars and karraginans are isolated, which are of broad usage as gelforming and thickening agents in nuclear engi neering, food, textile, lacqueranddye, and leather industries, as well as in microbiological and pharma ceutical processes [1, 2]. These marine organisms are reliable indicators of environment status. In addition, they can be used as natural biosorbents, properties of which, in the first order, are function of their extra cellular matrix [3–9]. It has been shown that up to 80% of the total amount of cadmium ions is accumu lated in the cell walls of Gracilaria cornea and only 20% is localized in the cell [8]. According to contemporary theories, the cell wall is a multifunctional system with complex organiza tion. It is an external compartment of a plant cell, which is the site of initial contact with external solu tions, and modifies its formula via the exchange reac tion between ionexchange groups of the cell wall polymeric matrix and ions in the environment. Effec tiveness of modification of the external solution by the cell wall is defined by its ionexchange properties, which play a crucial role in the absorption of sub stances by plants both under normal and extreme con ditions of the ionic environment (in particular, at high concentrations of heavy metals). There has been scarce research [8, 9] in the pecu liarities of functioning of the cell walls of red algae’s natural ionexchangers. Moreover, today there are no approaches to the evaluation of the composition of the algae’s cell wall ionexchange groups, which can ligate with ions of the external medium via exchange reac tions. The authors of [10, 11] have developed a meth odology of investigating the cell wall of high terrestrial plant roots without the use of methods of physical and chemical destruction, aimed at quantitative descrip tion of behavior of this natural ionexchanger and learning the peculiarities of its matrix’s participation in such complicated physiological process as absorp tion of minerals from the environment. The objective of the present paper is the study of the composition of ionogenic groups defining the ion exchange properties of the cell walls of a red alga, Phyl lophora crispa, which is a subdominant in upper areas of the phytal zone in the coastal biocenoses on the Black Sea [12]. METHODS Objects of study. The Black Sea red alga Phyllophora сrispa (Hudson) P.S. Dixon (=P. nervosa (DC.) Grev) was used. Thalluses were harvested by diving at the depth of 8 m near cape Malii Utrish. The collected material was dried at room temperature and cleared to a maximum possible extent from fouling of different origins. 176 IONEXCHANGE PROPERTIES OF CELL WALLS Isolation of the cell wall. The method earlier described for the roots of high plants [10, 11] was modified to a certain extent. The dried material was placed in a glass ionexchange column (V = 200 ml) and washed in dynamic conditions subsequently with 0.1% NaOH (approximately 0.5 l), deionized H2O, 1% HCl (approximately 0.5 l), then H2O until there was no Cl in the percolate. The detection of Cl ion was conducted via the titer method with mercury nitrate. Finally, the preparations were treated with alcohol and acetone and washed at room temperature. The obtained preparations are termed “standardized cell walls.” Cell wall quality estimate was controlled by the previously described method [10] using fluorescent microscopy (microscope Axioplan 2 imaging MOT, Carl Zeiss, Germany). Intrinsic cell structures were absent in the isolated cell walls, while the preparations fully retained the tissue architecture. Determination of ionexchange groups. We used the method of electrometric titers. Samples of dried cell wall preparations, 40 ± 0.1 mg each, were put in glazed weighing bottles with sealed cap (volume ~50 ml) and potted with solutions of NaOH or HCl (12.5 ml) in varying concentrations but with constant ionic strength, which was maintained with relevant solu tions of 10 mM NaCl. After 48 h, the samples were iso lated from the solution, dried with filter paper, and the mass of fluid material was defined G FCW . The samples were then dried at 50°С to constant weight, the dried mass was determined (G DCW ) and the weight coefficient of cell wall dwelling was computed. Also, we measured pH and concentrations of the acid or alkali in solu tions before and after contact with the samples via titration with methyl red. Sorption capacity of the cell wall at pHi was calculated based on the change of con centration of H+ or OH– using the formula: ( cat аn Si ( ) (C = ini ) )  C equi V g , (1) where Si is sorption capacity of samples for cations (Scat) or anions (San), µmol/g of the dried mass of the cell wall; Cini and Cequi are initial and relevant equilib rium concentrations of NaOH or HCl in the solution, mM; V is solution volume, ml; and g is weight of the sample, g. Titration curves Si = f(pHi) were computed using the previously developed method in accordance with differential curves (dSi/dpHi) = f(pHi) [10, 11]. The amount of groups in the cell wall of P. crispa was deter mined via the differential curves ( Δ S j ). In this case, the degree of ionization (αi) of the groups can be com puted using the following formula: j αi = Si j , ΔS APPLIED BIOCHEMISTRY AND MICROBIOLOGY (2) 177 where S i j is the amount of dissociated groups of the j type at pH i. To calculate the ionization constant for each iono genic group, we used Henderson–Hasselbach’s equa tion modified by Gregor [10]: pH = pK a + n log 10 (1 −αα), (3) where pK a is the seeming constant of ionization of the ionogenic group of the polymer, α is degree of ioniza tion, and n is constant depending on the structure of the polymeric matrix and nature of the counter ion [13]. If the dependence pH = f log 10 α is linear, 1−α then, in agreement with equation (3), the pH value at which the line intersects the y axis, will be equal to the value of pK a,, and the slope of the curve will match the value of the constant n (3). The value of S icalc was computed at the set values of parameters (ΔSj, pK aj, nj) [14]: ( ( )) k, m Sicalc = S Ocat − ∑ j, i = 1 ΔS 1 + 10 j ⎛ pK aj − pHi ⎞ ⎜ ⎟ nj ⎝ ⎠ , (4) S iрас where is the calculated ionexchange capacity of the cell wall at the relevant value of pHi; S Ocat is maxi mal cationexchange capacity of the cell walls; ΔS j is the amount of ionogenic groups of the jtype; S Ocat , ΔS j, and S icalc are expressed in µmol per 1 g of dried mass of the cell walls; pK aj is seeming constant of ionization of ionogenic groups of the jtype; nj is the constant of the equation (3) for ionogenic groups of the jtype; k is the amount of points on the electrometric curve; and mis the amount of types of ionogenic groups. Adequacy of the applied method for the descrip tion of acidicbase equilibrium was estimated by means of regression analysis with determination of parameters of the following equation: Sicalc = B ⋅ Siexp + A, (5) where and in µmol/g of dried mass of cell walls are experimental ionexchange capacity and ionexchange capacity computed from (4) at relevant pHi; A and B are regression parameters. Amino group content. To define the amount of amino groups in the cell wall polymeric matrix by the method of nonwater titration in the acetic acid [15], the sample of ground and dried cell wall preparation (20 mg) was potted with 7 ml, 10 mM solution of per chloric acid in glacial acetic acid. After 2 days, the samples were isolated from the solution. Prior to and after contact with the cell walls, the solution was titered with 10 mM potassium biphtalate solution in glacial acetic acid in the presence of crystal violet. The Vol. 47 Siexp No. 2 S icalc, 2011 178 MEICHIK et al. 800 RESULTS AND DISCUSSION S cat 600 400 200 0 2 4 6 8 10 –200 S аn 12 рН Fig. 1. Curve of electrometric titration of cell walls isolated from thallus of Phyllophora crispa. S, μmol/g of cell wall dried mass is ionexchange capacity of cell walls for cations (Scat, positive values) and anions (San, negative values). Firm line marks the calculated curve (equation 6); dots mean experimental data. Bars mean standard deviations. content of free amino groups ( N NH 2 ) was determined using the following formula [15]: N NH2 = (Vini − Vкон ) × N bpp × Vtotal (6) , Va × g where Vini and Vini is amount of potassium biphtalate which was consumed during the titration of the initial and final (after the contact with the preparations) solution, ml; Nbpp is normality of potassium biphta late, mM; Vα is quantity of the solution taken for titra tion, ml; Vtotal is total volume of the solution taken for potting the sample, ml; and g is mass of the sample, g. Weight coefficient of matrix dwelling. To define the weight coefficient of dwelling of the cell wall poly meric matrix in the water and solutions in the thallus of the seaweed, the standardized cell walls dwelt in water or solutions were dried with filter paper, and their fluid mass was determined (G Fcw ). Then, the cell wall samples were dried at room temperature, and their dried mass was defined (G Dcw ). The weight coeffi cient of dwelling of the standardized cell walls (Kcw) was calculated using the formula [16]: cw cw (7) K cw = G F −cwG D , GD where GF and GD is fluid and dried mass of the samples, g; and the index cw means “cell wall.” Definition of elements. The element analysis of the alga thallus and cell walls extracted from it was con ducted on the semiautomatic CHNS analyzer Per kinElmer 2400. Statistical analysis. Word processor Excel 7.0 was used. Figures used were mean values from 3–8 repeti tions and their standard deviations. The obtained results (Fig. 1) show that, in P. crispa, the cell walls have had both anion (San) and cation exchange (Scat) capacity. In water solutions, they were 80 and 700 µmol/g of dried mass of the cell walls, respectively. These data allow for a conclusion that, just as in high plants, red alga’s cell walls are a natural cationexchanger: they have predominantly cation exchange properties. However, the comparative analy sis of Scat and San indicates that the thallus’ call walls have 1.5–2 times as low sorption capacity both for anions and cations as sheathes isolated from roots of high plants [10]. The experimental curve of electrometric titer for the cell walls of P. crispa, as well as for polyfunctional ion exchangers, has had a complex, polysigmoid nature that evidences the presence of several types of functional groups in the polymeric matrix. Analysis of the above dependencies showed that the curves had three inflections that point at the presence of three functional group types in the structure of red alga’s cell walls. Thus, by the differential curves, we defined the amount of group types in the cell wall of P. crispa, as well as their amount ( Δ S j ). Using the values of αi (equation 2), the relevant values of pH, and the equa tion (3), for each ionogenic group, we calculated pK aj and nj. It should be noted that the equation (3) is suc cessfully used to describe the processes of acidicbase equilibrium both in the structure of polyfunctional synthetic ionexchangers [14, 17] and natural ion exchangers, to which the cell wall of plants belongs [10, 11, 18]. Calculations have shown that the selected model fully matches the experimental data obtained in the present study, which was indicated by values of correlation coefficients (rcorr) of dependencies ( ) exp (Fig. 2). S icalc = f S i Thus, in the structure of the polymeric matrix of the cell walls of P. crispa, three types of ionogenic groups that were able to participate in ionexchange reactions under relevant conditions were revealed. It is wellknown that sulfate and carboxyl groups are contained in red algae’s cell walls, within acidic polysaccharides. The data obtained from the element analysis evidence that the cell walls of P. crispa con tained 1.9% sulfur (Table 2). These results coincide with the current concepts that sulfated galactans are an important component in polysaccharides of red algae [19, 20]. However, the suggestion that the acidic groups revealed by us with рКа ~ 4.95 are sulfa groups runs counter to the obtained experimental results because these groups in the polymers have a character istic рКа ~ 1 [16]. It can be concluded on the basis of the abovesaid that alkylated sulfate groups are present in sulfated galactans of P. crispa, and their content reaches 600 µmol/g of dried mass of the cell wall (Table 2). It may be suggested that, in P. crispa, these APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 47 No. 2 2011 IONEXCHANGE PROPERTIES OF CELL WALLS groups engage in the formation of sulfone transverse connections between linear polysaccharide chains like those present in synthetic sulfurcontaining ion exchangers [21]. A comparison of values рКa obtained in this paper with the data on chemical composition of red algae’s cell walls [21], together with analysis of these values for different types of groups in lowmolecular compounds [22], has led us to the conclusion that the groups with рКa are carboxyl groups of uronic acids. The data harvested from the analysis of the cell wall and thallus of P. crispa indicate that not only sulfur containing but also nitrogencontaining polymers are an important component of its polymeric matrix, because the share of nitrogen in the walls has been 5.8% (Table 2). Talmadge et al. [23] evaluated protein content in the call wall polymeric matrix (Gpro) using data of total nitrogen analysis (Nkc) and performing calculations in accordance with the formula: Gpro = Nkc × 6.25. Similar calculations conducted for P. crispa show that the protein is the major component of the polymeric matrix because its share is 36% per 1 unit of dried mass of the cell wall. According to some authors [24], proteins in the cell walls of algae, as well as high plants, are glycoproteins which may contain up to 10% free amino groups. In agreement with our data, when titrating the polymeric matrix of the cell walls in the water medium in the range of pH < 3.5, no release of proton takes place; instead, it is absorbed (Fig. 1), i.e., in this pH range, the following reaction occurs: ∼RNH2 + HCl [RNH3]+Cl–, and the amount of the latter equals 80 µmol/g of the dried cell wall mass (Table 1). It may be assumed that the groups revealed in the said pH area are amino groups in the polymeric structure of the cell wall, because there are not any other major groups therein. For the cell walls of high plants, it was shown that, at titration in the water medium, not all amino groups in the polymeric matrix are determined due to the for mation of intrasalt form by free amino acid fragments (–СОО–NH3+ – ) in this matrix. To conduct complete identification, the nonwater titer method has to be used [15]. During application of the latter to the stan dardized preparations of the P. crispa cell walls, it was demonstrated that they contain 436 ± 24 micromoles of amino groups/g of dried mass of the cell walls, which is an over fivefold increase compared with that obtained with the water titers. These data suggest that, just as in high plants, free amino acid fragments are present in the polymeric matrix of red alga’s cell walls. This suggestion is supported by the following data. The amount of the groups with рКа ~7.7 we revealed is 420 µmol/g of dried mass of the cell walls, which, within the limits of experimental error, coincides with the total content of amino groups revealed by the nonwa ter titer method. Furthermore, one can assume that, with adding NaOH in the pH range 6–9 where the APPLIED BIOCHEMISTRY AND MICROBIOLOGY calc 800 S 179 y = 1.041x – 10.08 R2 = 0.994 600 400 200 S –200 0 200 400 600 exp 800 –200 Fig. 2. Coincidence between experimental and calculated data of electrometric titration of the cell wall of thallus of exp calc P. crispa. Si and Si , μmol/g of cell wall dried mass are experimental and calculated (equation 5) ionexchange capac ities of the cell wall of thallus of P. crispa at relevant value of pHi. The trend line equation is presented on the chart. proton’s release was observed, deprotonation of amino groups and, at the same time, the decay of Zwitter ionic form occur following the reaction: OOC–CH(R∼)–NH3+ + NaOH NaOOC–CH(R∼)–NH2 + H2O, – where R∼ is polymeric chain of the cell wall matrix. In accordance with the values рКа of the carboxyl group of amino acids (рКа ~2; [22]), protonation of the carboxyl group of the amino acid residue takes place at the titration with the acid and pH ≤ 3.5, the α ⎞ Table 1. Parameters of equation pH = pKα + n log ⎛  10⎝ 1 – α⎠ for cell walls of Phyllophora crispa at the ionic strength of the solution 10 mM NaCl j pK aj nj R 2j k ΔS j 1 2.88 ± 0.05 0.22 0.931 5 80 ± 10 2 4.60 ± 0.03 0.939 0.961 10 260 ± 15 3 7.67 ± 0.08 1.098 0.964 10 400 ± 20 j Note: j is group type; pK a is constant of ionization of the group of Vol. 47 jtype; n j is constant of equation for the group of jtype; ΔS j (μmol/g of cell wall dried mass) is the amount of groups of j type; k is the number of points on the line. 1—amino groups; 2—carboxyl groups of uronic acids; 3—cationexchange groups of the second type. Mean values and SD are presented. No. 2 2011 180 MEICHIK et al. Table 2. Element analysis of the thallus of Phyllophora crispa and cell wall isolated from it Algae material Thallus Cell wall N(I) C(I) H(I) S(I) N(II) S(II) 2.98 ± 0.12 5.77 ± 1.00 36.75 ± 0.59 41.37 ± 2.00 6.12 ± 0.02 6.69 ± 0.21 1.37 ± 0.19 1.92 ± 0.31 2.13 4.12 0.43 0.60 Note: Values N(I), C(I), H(I), and S(I) are given in percent, whereas N(II) and S(II), are given in mM of nitrogen and sulfur/g of dried mass of thallus and cell walls, respectively. Means from three analytical repetitions and their standard deviations are presented. I—data of element analysis; II—calculated values. Zwitterion form decays, and amino groups become positively charged: OOC–CH(R∼)–NH3+ + H+ НOOC–CH(R∼)–NH 3+. – It is for this reason that the anionexchange capacity of the cell wall displays only at the HCl concentration in the medium over 0.05 mM. The degree of ionization of weak acids and bases, which include ionogenic groups in the polymeric matrix of the cell walls of P. crispa, depends on two fac tors: on the values of pH and рКa. The latter is known to be constant for any acid or base. Consequently, for fixed pH value, the degree of ionization (α) will depend only on the nature of the acid (base) regardless of whether it is neutralized in advance [22]. The equa tion for calculating α has the following form: { } pH − pK a ) n] ⎤, (8) α = 1 ⎡⎣1 + 1 10[( ⎦ and can be used for the purpose of defining α of iono genic groups of the cell walls at the appropriate pH val ues in the external medium (Fig. 3). For example, at pH = 6, 94% carboxyl groups, having pKa ~5, is ion α 1.0 2 3 0.8 0.6 –COOH(1) 1 –COO–(1) 0.4 –COOH(2) 0.2 –NH3+ 0 2 –COO–(2) –NH2 4 6 8 10 pH Fig. 3. Dependence of degree of ionization (α) of different ionogenic groups in the thallus’ cell walls of P. crispa on pH of the external solution. Lines connect the calculated values obtained via equation (8) with the parameters from Table 1. 1—aminogroups; 2—carboxyl groups; 3—car boxyl groups of the second type. ized, whereas all carboxyl groups with pKa ~7.7 are not able to participate in the exchange reactions with cat ions of the external medium; at pH = 8, both groups are 100 and 65% ionized, respectively. It should be noted that amino groups are always nondissociated under physiological conditions (pH 4–9) and, hence, do not participate in exchange reactions with cations of the external medium (Fig. 3). Dwelling is one of important physicochemical indicators, which quantitatively characterize the properties of cell wall polymers. The quantitative fea ture of this process is the weight coefficient of dwell ing, being equal to the ratio of the amount of water in the polymer to 1 g of its dried mass. Dwelling of ion exchange substances in water solutions is caused by hydrophilic groups, while their insolubility is caused by the existence of transverse connections. The degree of ionexchange material’s dwelling depends on the properties of the ion exchanger and composition of the external medium. The dwelling capacity increases with decreasing degree of crosslinkage, increasing total amount of ionogenic groups, and rising their degree of dissociation, as well as decreasing solution concentration. Also, it depends on the radius of the hydrated ion which the adsorbent is filled with [16]. The results of the present study have shown that the value of dwelling factor varied from 1.5–1.9 g Н2О per unit of dried mass of cell walls in the range of pH 2–10 (Fig. 1) Proceeding from the knowledge of physico chemical laws of dwelling for synthetic ionexchang ers, one may assume that the main factor determining the dwelling capacity is the degree of crosslink of polymeric chains in the structure of the cell walls. This parameter can not be defined in experiments; how ever, there is a possibility to assess its value indirectly. In accordance with the data on the dwelling of cell walls of high plants [10] and the results of this paper, one may assume that, in P. crispa, the degree of cross link of polymeric chains in the cell walls is higher than in roots of the plants. This conclusion stems from the comparative analysis of the values of the dwelling coef ficient in solutions, which witnesses that, in red alga, this parameter is lower by 2–10 times than in the high plants. The obtained results show that, in contrast to the high plants, in P. crispa, the volume of the cell walls varies slightly and displays the low dependence on ion conditions and pH of the medium (Fig. 4). APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 47 No. 2 2011 IONEXCHANGE PROPERTIES OF CELL WALLS Kcw 4 3. 3 4. 2 1 5. 0 4 5 6 7 8 9 6. 10 pH 7. Fig. 4. Dependence of the dwelling coefficient (Кcw) of cell walls isolated from the thallus of P. crispa on pH of the external solution. Values of Кcw are expressed in g of H2O per 1 g of dried mass of the cell walls. Bars mean standard deviations. 8. 9. Thus, we have revealed that the ionexchange properties of the cell wall polymeric matrix of P. crispa are preconditioned by the presence of two types of cat ionexchange groups that are able to involve in exchange reactions with ions in the external medium. They are carboxyl groups of uronic acids and, proba bly, carboxyl groups of amino acid fragments. The dif ference in physicochemical properties of extracellu lar matrix of red algae and terrestrial high plants seems to rather reflect different life condition pathways of water and ion transfer from absorption sites to sites of utilization than depend on their taxonomic status. The developed approach to the quantitative assess ment of ionexchange properties of the cell wall poly meric matrix in P. crispa can be applied for defining analogous properties to other algae species. Taking into account that it is the extracellular matrix plays the role of the adsorbent due to mechanisms of ion exchange, quantitative indicators, which feature sorp tion properties of the cell walls of other types of algae, can be used for their selection for ecological monitor ing and for biosorption. The present study was performed at financial sup port of the Russian Foundation for Basic Research, grants 040449379a and 080401398a, and the Fed eral Target Program “Scientific and ScientificPedagog ical Cadre of the Innovative Russia,” in the field “Cellu lar Technologies” (State Contract no. P403). 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. REFERENCES 1 2 22. 1. Maksimova, O.V., in Biologiya chernomorskikh agarofi tov (Biology of BlackSea Agarophytes), Moscow: IORAN, 1993, pp. 7–24. 23. 2. Vozzhinskaya, V.B. and Kamnev, A.N., Ekologobiolog icheskie osnovy kul’tivirovaniya i ispol’zovaniya mor skikh donnykh vodoroslei (Ecological and Biological 24. APPLIED BIOCHEMISTRY AND MICROBIOLOGY SPELL: 1. Maksimova, 2. Kamnev, 3. Burdin View publication stats Vol. 47 181 Basics of Cultivation and Use of Marine Benthic Algae), Moscow: Nauka, 1994. Burdin, K.S., Osnovy ekologicheskogo monitoringa 3 (Fundamentals of Ecological Monitoring), Moscow: Mosk. Gos. Univ., 1985. Kamnev, A.N., Voskoboinikov, G.M., Vershinin, A.O., 2 Zhil’tsova, L.V., and Vozzhinskaya, V.B., in Biologiya chernomorskikh agarofitov (Biology of BlackSea Agar ophytes), Moscow: IORAN, 1993, pp. 141–162. Topcuoglu, S., Guven, K.C., Balks, N., and Krbasoglu, C., Chemosphere, 2003, vol. 52, no. 10, pp. 1683–1688. Vilar, V.J.P., Botelho, C.M.S., and Boaventura, R.A.R., Water Res., 2006, vol. 40, no. 2, pp. 291–302. Murphy, V., Hughes, H., and McLoughlin, P., Water Res., 2007, vol. 41, no. 4, pp. 731–740. GarciaRios, V., FreilePelegrin, Y., Robledo, D., MendozaCozatl, D., MorenoSanchez, R., and GoldBouchot, G., Aquatic Toxicol., 2007, vol. 81, no. 1, pp. 65–72. Herrero, R., Lodeiro, P., Rojo, R., Ciorba, A., Rod riguez, P., and Sastre de Vicente, M.E., Bioresource Technol., 2008, vol. 99, no. 10, pp. 4138–4146. Meychik, N.R. and Yermakov, I.P., Plant Soil, 2001, vol. 234, no. 2, pp. 181–193. Meychik, N.R., Nikolaeva, J.I., and Yermakov, I.P., Plant Soil, 2005, vol. 277, nos. 12, pp. 163–174. KaluginaGutnik, A.A., Fitobentos Chernogo morya (Phytobenthos of the Black Sea), Kiev: Naukova dumka, 1975. Shataeva, L.K., Kuznetsova, N.N., and El’kin, G.E., Karboksil’nye kationity v biologii (Carboxyl Cationites in Biology), Leningrad: Nauka, 1979. Leikin, Yu.A., Meychik, N.R., and Solov’ev, V.K., Zh. Fiz. Khim., 1978, vol. 52, no. 7, pp. 1420–1424. Meychik, N.R., Nikolaeva, Yu.I., and Yermakov, I.P., Biokhimiya, 2009, vol. 74, no. 8, pp. 1145–1151. Gel’ferikh, F., Ionity (Ionites), Moscow: Inostr. Liter atura, 1962. Meichik, N.R., Leikin, Yu.A., and Geinrikh, I.A., Zh. Fiz. Khim., 1989, vol. 63, no. 8, pp. 2164–2168. Meychik, N.R. and Yermakov, I.P., Plant Soil, 1999, vol. 217, nos. 12, pp. 257–264. Kloareg, B. and Quatrano, R., Oceanorg. Mar. Biol. Ann. Rev., 1988, vol. 26, pp. 259–315. Usov, A.I., in Morskie pribrezhnye ekosistemy: vodorosli, bespozvonochnye i produkty ikh pererabotki. Mater. II mezhd. nauchnoprakticheskoi konf. (Marine Coastal Ecosystems: Algae, Invertebrates, and the Products of Their Processing. Proc. II Int. Sci.Pract. Conf.), Mos cow: Izd. VNIRO, 2002, pp. 38–52. Tverskoi, V.A., Fedotov, Yu.A., Vdovin, P.A., and Dub yaga, V.P., Membrany, Ser. Krit. Tekhnol., 2007,m vol. 36, no. 1, pp. 17–40. Albert, A. and Serjeant, E., Ionization Constants, Lon don: Methuen, 1962. Talmadge, K.W., Keegstra, K., Bauer, W.D., and Alber sheim, P., Plant Physiol., 1973, vol. 51, no. 1, pp. 158– 173. Gassab, G.I., Annu. Rev. Plant Physiol. Plant Mol. Biol., 1998, vol. 49, pp. 281–309. No. 2 2011